Extracellular vesicles from subjects with COPD modulate cancer initiating cells phenotype through HIF-1α shuttling

Chronic obstructive pulmonary disease (COPD) is a risk factor for lung cancer development. COPD induces activation of hypoxia-induced signaling, causing remodeling of surrounding microenvironmental cells also modulating the release and cargo of their extracellular vesicles (EVs). We aimed to evaluate the potential role of circulating EVs from COPD subjects in lung cancer onset. Plasma-EVs were isolated by ultracentrifugation from heavy smoker volunteers with (COPD-EVs) or without (heavy smoker-EVs, HS-EV) COPD and characterized following MISEV guidelines. Immortalized human bronchial epithelial cells (CDK4, hTERT-HBEC3-KT), genetically modified with different oncogenic alterations commonly found in lung cancer (sh-p53, KRASV12), were used to test plasma-EVs pro-tumorigenic activity in vitro. COPD-EVs mainly derived from immune and endothelial cells. COPD-EVs selectively increased the subset of CD133+CXCR4+ metastasis initiating cells (MICs) in HBEC-sh-p53-KRASV12high cells and stimulated 3D growth, migration/invasion, and acquisition of mesenchymal traits. These effects were not observed in HBEC cells bearing single oncogenic mutation (sh-p53 or KRASV12). Mechanistically, hypoxia-inducible factor 1-alpha (HIF-1α) transferred from COPD-EVs triggers CXCR4 pathway activation that in turn mediates MICs expansion and acquisition of pro-tumorigenic effects. Indeed, HIF-1α inhibition or CXCR4 silencing prevented the acquisition of malignant traits induced by COPD-EVs alone. Hypoxia recapitulates the effects observed with COPD-EVs in HBEC-sh-p53-KRASV12high cells. Notably, higher levels of HIF-1α were observed in EVs from COPD subjects who subsequently developed cancer compared to those who remained cancer-free. Our findings support a role of COPD-EVs to promote the expansion of MICs in premalignant epithelial cells through HIF-1α-CXCR4 axis activation thereby potentially sustaining lung cancer progression.

For hypoxia experiments, cells were maintained in a Whitley H25 Hypoxystation (Don Whitley Scientific Limited, UK) with 2% oxygen at 37 °C for 48 h.
In experiments where HBEC-KRAS V12high were treated with EVs obtained from the conditioned medium of HUVEC cells kept in normoxic or hypoxic condition, 10 µg of EVs were used.

EVs characterization
NanoSight: The EVs concentration and the size distribution were evaluated by using a NanoSight NS300 instrument (Malvern Panalytical).Videos obtained were analyzed using NTA 3.2 software.The morphology of EVs was determined using a Zeiss LIBRA 200FE transmission electron microscope with an in-column second-generation Omega filter.The size of EVs was quantified by analyzing 100 EVs using the iTEM imaging platform.
MACSPlex analysis: To determine the surface marker profile of plasma EVs, MACSPlex Kit (Milteny Biotec, Bergisch-Gladbach, Germany) was used following manufacturer's instructions.Briefly, 15 µg of EVs were incubated with MACSPlex Exosome Capture Beads which are fluorescently labeled beads coated with 37 different antibodies that recognize corresponding surface epitopes on EVs.Epitopes recognized by beads are listed below.

Flow Cytometry for HIF-1α analysis
To evaluate the transfer of HIF-1α carried by EVs into recipient cells, HBEC-KRAS V12high cells were treated for 24 h with 15 µg of PKH26-labelled-EVs.Then, cells were collected and fixed with the Fixation Buffer (BD Bioscience) for 30 min at 4 °C.After the fixation, cells were permeabilized with the Permeabilization buffer (eBioscience) and stained with AF 405-conjugate anti-human HIF-1α antibody (clone: 241812 cat.n: #IC1935V-100UG, R&D SYSTEMS) for 30 min at 4 °C.After the staining, cells were washed with TF PERM/WASH buffer 5X (eBioscience) and the acquisition was performed using a FACSCanto II flow cytometer.All flow cytometry analyses were performed by FlowJo software.

Flow Cytometry for EMT marker
To assess if COPD-and HS-EVs treatment could affect the expression of markers involved in the EMT process in HBEC-KRAS V12high cells, the expression of CDH1, CHD2, ITGA6, FN1 and VIM were analyzed by flow cytometry.For CDH1, CHD2, ITGA6 staining, cells were collected and stained with antibodies of interest, reported in the List of antibodies, for 20 min at 4 °C.After washing, cells were incubated with 7-AAD live/dead solution (Thermo Fisher Scientific) prior to the acquisition.For the intracellular staining of FN1 and VIM, the protocol reported for HIF-1α flow cytometry experiments was performed as reported above, but using antibodies of interest, reported in the List of antibodies.Samples were acquired using a FACSCanto II flow cytometer.All flow cytometry analyses were performed by FlowJo software.

Western Blot
EVs (30 µg) were incubated with RIPA buffer and protease inhibitors cocktail for 30 min on ice.Protein samples were then run on a 4-12 % polyacrylamide gel (Thermo Fisher Scientific) and transferred to a nitrocellulose transfer membrane using the iBlot 2 Gel Transfer Device (Thermo Fisher Scientific).Aspecific binding sites were blocked by incubating membranes with 5 % non-fat milk in T-TBS 1X.After the blocking, membranes were incubated overnight with primary antibodies of interest.For this study, primary antibodies used with their dilution were: rabbit anti-human CD9 monoclonal antibody 1:1000 (cat.n: 74220, Cell Signaling Technology, Danvers, Massachusetts, USA), rabbit anti-human CD81 monoclonal

Migration and Invasion Assay
For the migration assay, 5x10 4 cells were plated into a 6 well plate and treated with 15 µg of HS-EVs or COPD-EVs.After 24 h, 1x10 5 of treated cells were resuspended in 100 µl of RPMI free medium and then seeded into the top chamber of FluoroBlok 24 well cell culture inserts with 8 µm pore size (Corning, Glandale, AZ, USA).Instead, in the lower chamber, 750 µl of Keratynocite-SFM with EGF (Epithelial Grow Factor 100 ng/ml) or SDF-1 (50 ng/ml) was added.After 24 h, migrated cells were fixed with methanol 30% and their nuclei stained with VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories, Newark, CA, USA).The numbers of migrated cells (3 random fields/condition) were counted through fluorescence microscopy.For the invasion assay, inserts were coated with Matrigel (Becton Dickinson, Franklin Lakes, NJ, USA) and the assay was stopped after 48 h.
antibody 1:1000 (cat.n: 10037, Cell Signaling Technology), rabbit anti-human Tsg101 monoclonal antibody 1:1000 (cat.n: 72312, Cell Signaling Technology) and mouse anti-human ApoA1 monoclonal antibody 1:1000 (cat.n. number: 3350, Cell Signaling Technology).After 3 washing with T-TBS 1X, membranes were incubated with HRP-conjugated goat anti-rabbit IgG antibody 1:5000 (cat n: 7074, Cell Signaling Technology) or HRP-conjugated goat anti-mouse antibody 1:5000 (cat n: 31340, GE Healthcare Life Sciences, USA).Signals were detected via an enhanced chemiluminescence reaction (GE Healthcare Life Sciences, USA) in a MINI HD9 Western Blot Imaging System (Cleaver Scientific, UK).Images of original western blots for CD9, CD81, TSG101 and ApoA1 are shown in Supplemental Fig.S2A-DFluorescence membrane staining of EVs for uptake experiments HS-EVs and COPD-EVs (15 µg) were labeled with the red fluorescence dye PKH26 (Sigma-Aldrich), a lipophilic dye used for the general labeling of cell membrane.Briefly, EVs were incubated with 1 μl of PKH26 dye diluted in 1 ml PBS for 5 min at RT.The dye excess was removed by washing EVs by ultracentrifugation at 120 000 × g for 60 min at 4 °C.Finally, labeled EVs were resuspended in filtered PBS and stored at -80 °C as 1 µg/aliquots.